We are engaged in analysis of the lytic replication of two human herpesviruses: herpes simplex virus (HSV) and Epstein-Barr virus (EBV). We are studying the purified protein products of the viral genes that participate in DNA replication in lytically-infected cells using both biochemical and molecular genetic approaches to understand the function of these polypeptides in detail. Our recent results regarding HSV proteins can be summarized as follows: 1) UL9 ,the viral protein that presumably initiates DNA replication, interacts specifically with ICP8, the virus-encoded, single-stranded DNA binding protein; 2) the products of the HSV genes UL5, UL8, and UL52 form a three-polypeptide complex that has both helicase and primase activities. We have mapped the regions of UL52 that are essential for interaction with UL5 and UL8 and have obtained preliminary evidence that UL52 is essential for primase catalysis; 3) the HSV DNA polymerase consists of a stable complex of two polypeptides: UL30 (the catalytic subunit) and UL42 (an accessory subunit that increases the processivity of the enzyme). Our results have shown that the increase in processivity brought about by UL42 does not result in an increase in the fidelity of DNA synthesis; 4) we have reconstituted rolling-circle DNA replication in vitro using purified HSV replication proteins. Since this reactions depends on six of the seven viral gene products required in vivo, we are exploiting this system to gain insight into how these polypeptides interact at a replication fork. In addition, we have developed an assay to investigate the involvement of cellular proteins in viral DNA replication.